The goal of this research is to analyze the cellular and molecular mechanisms which enable nerve fibers to grow accurately to their targets in the developing embryo. A simple model system, peripheral pioneer neurons in insect embryonic limb buds, will be investigated. As many key extracellular, cell surface, cytoplasmic, and cytoskeletal molecules involved in this process are highly homologous in insects and vertebrates, the results obtained at the molecular and cellular level in this system are excellent models of the corresponding processes in vertebrates. These processes of cell recognition and directed motility are fundamental in the development of all cellular systems in all organisms; achieving an adequate understanding of the mechanisms involved will have a great impact on our ability to recognize, and alleviate, developmental disorders. Directed nerve cell outgrowth involves: (1) ordered disposition of cellular and molecular guidance cues in the embryonic environment encountered by the extending fiber, (2) neural cell surface mechanisms for detecting guidance information, and (3) intracellular mechanisms which reorient or steer the construction of the neural cytoskeleton in response to detected guidance information. The advantage of the insect system in analysis of this problem is the combination of powerful molecular genetics, detailed information on the cellular environment, and ready observational and experimental access to intracellular events in identified neurons growing on their normal, in vivo, cellular environment. The specific goals of this project period are: (1) identify and characterize substrate and neuron cell surface molecules involved in guidance; (2) in identified neural growth cones migrating on the in vivo substrate, characterize the mechanism of selective microtubule assembly during steering events; (3) in identified neural growth cones migrating on the in vivo substrate, determine the role of cytoplasmic calcium ion concentration as a possible intracellular signaling mechanism during steering events.